As the human genome comes close to being completely identified, attention is turning to protein characterization, because the proteins are the substances actually involved in physiological processes, individually, posttranslationally modified and/or as part of complexes. State-of-the-art protein identification is based on mass spectrometric characterization of proteolytic peptides, either through peptide mapping data or through sequence tag data, combined with data base searching. In addition to protein identification, identification of post-translational modifications and sites of modification are extremely important aspects of protein characterization. Phosphorylation of proteins controls basic cellular processes and determination of the state of phosphorylation of a protein and the specific sites of phosphorylation provides important information that can be used for proposing mechanistic hypotheses, as well as for understanding the processes involved. We are actively developing and applying mass spectrometry-based methods for the characterization of phosphoproteins.